Interferon inducing genetically engineered attenuated viruses

ABSTRACT

The present invention relates to genetically engineered attenuated viruses and methods for their production. In particular, the present invention relates to engineering live attenuated viruses which contain a modified NS gene segment. Recombinant DNA techniques can be utilized to engineer site specific mutations into one or more noncoding regions of the viral genome which result in the down-regulation of one or more viral genes. Alternatively, recombinant DNA techniques can be used to engineer a mutation, including but not limited to an insertion, deletion, or substitution of an amino acid residue(s) or an epitope(s) into a coding region of the viral genome so that altered or chimeric viral proteins are expressed by the engineered virus.

[0001] The work reflected in this application was supported, in part, bya grant from the National Institutes of Health, and the Government mayhave certain rights in the invention.

1. INTRODUCTION

[0002] The present invention relates to engineering attenuated virusesby altering a non-coding region or the coding sequence of a viralnonstructural (NS) gene. In particular, the present invention relates toengineering live attenuated influenza viruses which induce interferonand related pathways. The present invention further relates to the useof the attenuated viruses and viral vectors against a broad range ofpathogens and/or antigens, including tumor specific antigens. Thepresent invention also relates to a host-restriction based selectionsystem for the identification of genetically manipulated influenzaviruses. In particular, the present invention relates to a selectionsystem to identify influenza viruses which contain modified NS genesegments.

2. BACKGROUND OF THE INVENTION 2.1. Attenuated Viruses

[0003] Inactivated virus vaccines are prepared by “killing” the viralpathogen, e.g., by heat or formalin treatment, so that it is not capableof replication. Inactivated vaccines have limited utility because theydo not provide long lasting immunity and, therefore, afford limitedprotection. An alternative approach for producing virus vaccinesinvolves the use of attenuated live virus vaccines. Attenuated virusesare capable of replication but are not pathogenic, and, therefore,provide for longer lasting immunity and afford greater protection.However, the conventional methods for producing attenuated virusesinvolve the chance isolation of host range mutants, many of which aretemperature sensitive; e.g., the virus is passaged through unnaturalhosts, and progeny viruses which are immunogenic, yet not pathogenic,are selected.

[0004] Recombinant DNA technology and genetic engineering techniques, intheory, would afford a superior approach to producing an attenuatedvirus since specific mutations could be deliberately engineered into theviral genome. However, the genetic alterations required for attenuationof viruses are not known or predictable. In general, the attempts to userecombinant DNA technology to engineer viral vaccines have mostly beendirected to the production of subunit vaccines which contain only theprotein subunits of the pathogen involved in the immune response,expressed in recombinant viral vectors such as vaccinia virus orbaculovirus. More recently, recombinant DNA techniques have beenutilized in an attempt to produce herpes virus deletion mutants orpolioviruses which mimic attenuated viruses found in nature or knownhost range mutants. Until very recently, the negative strand RNA viruseswere not amenable to site-specific manipulation at all, and thus couldnot be genetically engineered.

2.2. The Influenza Virus

[0005] Virus families containing enveloped single-stranded RNA of thenegative-sense genome are classified into groups having non-segmentedgenomes (Paramyxoviridae, Rhabdoviridae) or those having segmentedgenomes (Orthomyxoviridae, Bunyaviridae and Arenaviridae). TheOrthomyxoviridae family, described in detail below, and used in theexamples herein, contains only the viruses of influenza, types A, B andC.

[0006] The influenza virions consist of an internal ribonucleoproteincore (a helical nucleocapsid) containing the single-stranded RNA genome,and an outer lipoprotein envelope lined inside by a matrix protein (M).The segmented genome of influenza A consists of eight molecules (sevenfor influenza C) of linear, negative polarity, single-stranded RNAswhich encode ten polypeptides, including: the RNA-directed RNApolymerase proteins (PB2, PB1 and PA) and nucleoprotein (NP) which formthe nucleocapsid; the matrix proteins (M1, M2); two surfaceglycoproteins which project from the lipoprotein envelope: hemagglutinin(HA) and neuraminidase (NA); and nonstructural proteins whose functionis unknown (NS1 and NS2). Transcription and replication of the genometakes place in the nucleus and assembly occurs via budding on the plasmamembrane. The viruses can reassort genes during mixed infections.

[0007] Influenza virus adsorbs via HA to sialyloligosaccharides in cellmembrane glycoproteins and glycolipids. Following endocytosis of thevirion, a conformational change in the HA molecule occurs within thecellular endosome which facilitates membrane fusion, thus triggeringuncoating. The nucleocapsid migrates to the nucleus where viral mRNA istranscribed as the essential initial event in infection. Viral mRNA istranscribed by a unique mechanism in which viral endonuclease cleavesthe capped 5′-terminus from cellular heterologous mRNAs which then serveas primers for transcription of viral RNA templates by the viraltranscriptase. Transcripts terminate at sites 15 to 22 bases from theends of their templates, where oligo(U) sequences act as signals for thetemplate-independent addition of poly(A) tracts. Of the eight viral mRNAmolecules so produced, six are monocistronic messages that aretranslated directly into the proteins representing HA, NA, NP and theviral polymerase proteins, PB2, PB1 and PA. The other two transcriptsundergo splicing, each yielding two mRNAs which are translated indifferent reading frames to produce M1, M2, NS1 and NS2. In other words,the eight viral mRNAs code for ten proteins: eight structural and twononstructural. A summary of the genes of the influenza virus and theirprotein products is shown in Table I below. TABLE I INFLUENZA VIRUSGENOME RNA SEGMENTS AND CODING ASSIGNMENTS Length_(d) MoleculesLength_(b) Encoded (Amino Per Segment (Nucleotides) Polypeptide_(c)Acids) Virion Comments 1 2341 PB2 759 30-60 RNA transcriptase component;host cell RNA cap binding 2 2341 PB1 757 30-60 RNA transcriptasecomponent; initiation of transcription; endonuclease activity? 3 2233 PA716 30-60 RNA transcriptase component; elongation of mRNA chains? 4 1778HA 566  500 Hemagglutinin; trimer; envelope glycoprotein; mediatesattachment to cells 5 1565 NP 498 1000 Nucleoprotein; associated withRNA; structural component of RNA transcriptase 6 1413 NA 454  100Neuraminidase; tetramer; envelope glycoprotein 7 1027 M₁ 252 3000 Matrixprotein; lines inside of envelope M₂ 96 Structural protein in plasmamembrane; spliced mRNA ? ?9 Unidentified protein 8 890 NS₁ 230Nonstructural protein; function unknown NS₂ 121 Nonstructural protein;function unknown; spliced mRNA

[0008] The Influenza A genome contains eight segments of single-strandedRNA of negative polarity, coding for nine structural and onenonstructural proteins. The nonstructural protein NS1 is abundant ininfluenza virus infected cells, but has not been detected in virions.NS1 is a phosphoprotein found in the nucleus early during infection andalso in the cytoplasm at later times of the viral cycle (King et al.,1975, Virology 64: 378). Studies with temperature-sensitive (ts)influenza mutants carrying lesions in the NS gene suggested that the NS1protein is a transcriptional and post-transcriptional regulator ofmechanisms by which the virus is able to inhibit host cell geneexpression and to stimulate viral protein synthesis. Like many otherproteins that regulate post-transcriptional processes, the NS1 proteininteracts with specific RNA sequences and structures. The NS1 proteinhas been reported to bind to different RNA species including: vRNA,poly-A, U6 _(sn)RNA, 5′ untranslated region as of viral mRNAs and ds RNA(Qiu et al., 1995, Rna 1: 304; Qiu et al., 1994, J. Virol. 68: 2425).Expression of the NS1 protein from cDNA in transfected cells has beenassociated with several effects: inhibition of nucleo-cytoplasmictransport of mRNA, inhibition of pre-mRNA splicing, inhibition of hostmRNA polyadenylation and stimulation of translation of viral mRNA(Fortes, et al., 1994, Embo J. 13: 704; Enami, K. et al, 1994, J. Virol.68: 1432 de la Luna, et al., 1995, J. Virol. 69:2427; Lu, Y. et al.,1994, Genes Dev. 8:1817; Park, et. al., 1995, J. Biol Chem. 270, 28433).

3. SUMMARY OF THE INVENTION

[0009] The present invention relates to genetically engineered liveattenuated viruses which induce an interferon and related responses. Ina preferred embodiment the present invention relates to engineering liveattenuated influenza viruses which contain modified NS gene segments.The present invention also relates to both segmented and non-segmentedviruses genetically engineered to have an attenuated phenotype and aninterferon inducing phenotype, such a phenotype is achieved by targetingthe viral gene product which interferes with the cellular interferonresponse. The attenuated viruses of the present invention may beengineered by altering the non-coding region of the NS gene segment thatregulates transcription and/or replication of the viral gene so that itis down regulated. In non-segmented viruses, the down regulation of aviral gene can result in a decrease in the number of infectious virionsproduced during replication, so that the virus demonstrates attenuatedcharacteristics. A second approach involves engineering alterations ofthe NS coding region so that the viral protein expressed is altered bythe insertion, deletion or substitution of an amino acid residue or anepitope and an attenuated chimeric virus is produced. This approach maybe applied to a number of different viruses and is advantageously usedto engineer a negative strand RNA virus in which a NS gene product playsa role in regulating the interferon-mediated inhibition of translationof viral proteins.

[0010] The present invention is further related to vaccines and methodsof inhibiting viral infection. The attenuated viruses of the presentinvention may be used to protect against viral infection. Asdemonstrated by the evidence presented in the Examples herein, theattenuated viruses of the present invention have anti-viral activitywhen administered prior to infection with wild-type virus, thusdemonstrating the prophylactic utility of the attenuated viruses of thepresent invention.

[0011] The present invention is further related to a host-restrictionbased selection system for the identification of genetically manipulatedinfluenza viruses. The selection system of the present invention is moreparticularly related to the identification of genetically manipulatedinfluenza viruses which contain modified NS gene segments.

[0012] The present invention is based, in part, on the Applicants'surprising discovery that an engineered influenza A virus deleted of theNS1 gene was able to grow in a cell line deficient in type 2 IFNproduction, but was undetectable in Madin-Darby canine kidney (MDCK)cells and in the allantoic membrane of embryonated chicken eggs, twoconventional substrates for influenza virus. The Applicants' furtherdiscovered that the infection of human cells with the engineeredinfluenza A virus deleted of the NS1 gene, but not the wild-type virus,induced high levels of expression of genes under control of IFN-inducedpromoter. These results allow for the first time an efficient selectionsystem for influenza viruses which contain NS1 mutants, where previouslyit was not possible to screen for viruses with an NS1 deleted phenotype.

[0013] The attenuated viruses of the invention may advantageously beused safely in live virus vaccine formulation. As used herein, the term“attenuated” virus refers to a virus which is infectious but notpathogenic; or an infectious virus which may or may not be pathogenic,but which either produces defective particles during each round ofreplication or produces fewer progeny virions than does thecorresponding wild type virus during replication. Pathogenic viruseswhich are engineered to produce defective particles or a reduced numberof progeny virions are “attenuated” in that even though the virus iscapable of causing disease, the titers of virus obtained in a vaccinatedindividual will provide only subclinical levels of infection.

4. DESCRIPTION OF THE FIGURES

[0014]FIG. 1. Schematic representations of the NS genes and NS-specificmRNAs of (A) wild-type influenza A/PR/9/34 virus (WT NS) and (B)transfectant delNS1 influenza virus. Genomic RNA segments arerepresented as white boxes flanked by black squares. The latterrepresent the non coding regions of the gene. NS-specific mRNAs are alsorepresented. Thin lines at the ends of the mRNAs represent untranslatedregions. 5′ cap structures (black circles) and poly(A) tails in themRNAs are shown. The open reading frame of the NS1 protein isrepresented as a grey box. The specific-NEP (Nuclear Export Protein)open reading frame is shown as a hatched box. The NEP mRNA derived fromthe wild-type NS gene is a spliced product of the NS1 mRNA, as indicatedby the V-shaped line.

[0015]FIG. 2. RT-PCR analysis of the NS RNA segment of delNS1transfectant virus. The NS viral RNA from purified influenza A/PR/8/33virus (wt) or from delNS1 virus (delNS1) was amplified by coupledreverse transcription-PCR using the oligonucleotide primers described inSection 6. The PCR products were run on a 2% agarose gel and stainedwith ethidium bromide. The positions of size markers are indicated onthe right.

[0016]FIG. 3. Protein expression in delNS1 virus-infected (A) Vero cellsand (B) MDCK cells. Cells were infected with delNS1 virus at an MOI of0.02, [³⁵S] labeled at the indicated time points, and total amount ofviral proteins was immunoprecipitated using a polyclonal antiserumagainst influenza virus. Immunoprecipitated products were analyzed bySDS-PAGE. The major structural viral proteins, hemagglutinin (HA),nucleoprotein (NP), neuraminidase (NA) and matrix protein (Ml) areindicated by the arrows. Molecular size markers are shown on the left.

[0017]FIG. 4. Protein expression in delNS1 virus-infected IFNαR−/−cells. Cells were infected with delNS1 virus at an MOI of 0.02, and[³⁵S] labeled at the indicated time points. As a control, delNS1virus-infected Vero cells were labeled in the same experiment from 8 to10 h postinfection. Total amount of viral proteins wasimmunoprecipitated using a polyclonal antiserum against influenza virus.Immunoprecipitated products were analyzed by SDS-PAGE. The majorstructural viral proteins, hemagglutinin (HA), nucleoprotein (NP),neuraminidase (NA) and matrix protein (Ml) are indicated by the arrows.Molecular size markers are shown on the left.

[0018]FIG. 5. Induction of transcription from an IFN-stimulated promoterby infection with delNS1 virus. 293 cells were transfected with plasmidpHISG54-1-CAT encoding the reporter gene CAT under the control of a typeI IFN-stimulated promoter. One day posttransfection, cells weretransfected with 50 μg of dsRNA, or infected with delNS1 virus or withwild-type influenza A/PR/8/34 virus (wt) at the indicated MOIs. One daypostinfection, CAT activity was determined in cell extracts. Thestimulation of CAT activity following thy different treatments isindicated.

[0019]FIG. 6. Induction of antiviral response in embryonated eggs bydelNS1 virus. 10-day old embryonated chicken eggs were inoculated with20,000 plaque forming units of delNS1 virus or with PBS (untreated).After 8 h incubation at 37° C., the eggs were reinfected with 10³ pfu ofH1N1 influenza A/WSN/33 virus (WSN), H1N1 influenza A/PR/8 virus (PR8),H3N2 influenza A/X-31 virus (X-31), influenza B/Lee/40 virus (B-Lee), orSendai virus (Sendai). B-Lee infected eggs were incubated at 35° C. foradditional 40 h. All other eggs were incubated at 37° C. for additional40 h. Virus present in the allantoic fluid was titrated byhemagglutination assay.

5. DETAILED DESCRIPTION OF THE INVENTION

[0020] The present invention relates to genetically engineeredattenuated viruses and methods for their production. In particular, thepresent invention relates to engineering live attenuated viruses whichcontain a modified NS gene segment. Recombinant DNA techniques can beutilized to engineer site specific mutations into one or more noncodingregions of the viral genome which result in the down-regulation of oneor more viral genes. Alternatively, recombinant DNA techniques can beused to engineer a mutation, including but not limited to an insertion,deletion, or substitution of an amino acid residue(s) or an epitope(s)into a coding region of the viral genome so that altered or chimericviral proteins are expressed by the engineered virus.

[0021] The present invention further relates to a novel selection systemto identify influenza viruses containing a modified NS gene segment. Theselection system of the present invention is based, in part, on thehost-restriction of wild-type influenza virus and the ability ofinfluenza virus carrying a modification in the NS gene segment to infectand grow in an IFN-deficient cell.

[0022] The present invention is based, in part, on the Applicants'surprising discovery that an engineered influenza A virus deleted of theNS1 gene segment is able to grow in a cell line deficient in IFNproduction, but is undetectable in Madin-Darby canine Kidney (MDCK)cells and in the allantoic membrane of embryonated chicken eggs, twoconventional substrates for influenza virus. The engineered influenzavirus deleted of NS1 was further found by Applicants to induce IFNresponses in human cells. The Applicants also found that an engineeredinfluenza viruses deleted of NS1 is capable of replicating and inducingdisease in animals that were deficient in IFN signaling, but isnonpathogenic in wild-type mice.

[0023] The present invention further relates to the use of theattenuated viruses of the present invention as a vaccine against a broadrange of viruses and/or antigens, including tumor specific antigens.Many methods may be used to introduce the live attenuated virusformulations to a human or animal subject to an immune response. Theseinclude, but are not limited to, oral, intradermal, intramuscular,intraperitoneal, intravenous, subcutaneous and intranasal routes. In apreferred embodiment, the attenuated viruses of the present inventionare formulated for delivery as an intranasal vaccine.

[0024] 5.1. Attenuated Viruses which Induce Interferon Responses

[0025] The present invention relates to genetically engineered negativestrand RNA viruses containing a modification, mutation, substitution, ordeletion in the gene whose product is responsible for the virus bypassof the cellular interferon response. Thus, the present invention relatesto genetically engineered RNA viruses, both segmented and non-segmented,containing a mutation in the gene responsible for down-regulating thecellular IFN response. The genetically engineered attenuated viruses ofthe present invention have an interferon-inducing phenotype, as opposedto the wild-type viruses which inhibit cellular interferon mediatedresponses.

[0026] In a preferred embodiment, the present invention relates toattenuated influenza viruses with a modified NS gene segment and methodsof identifying those modified influenza viruses. The present inventionis based, in part, on the discovery that although a NS1 modified virusis able to grow in IFN deficient cells, such as, Vero cells, its abilityto replicate was severely impaired in MDCK cells and in embryonatedchicken eggs. It could be possible that these growth deficiencies aredue to changes in RNA segments other than the NS gene. In order to ruleout this possibility, the virus was “repaired” by rescuing an engineeredwild-type NS gene into the delNS1 virus. The resulting transfectantvirus grew to wild-type levels in MDCK cells and in eggs, demonstratingthat the lack of the NS1 gene determines the phenotypic characteristicsof the delNS1 virus.

[0027] Since NS modified viruses are capable of replicating in Verocells which are deficient in IFN expression, this indicates that alteredtissue culture and egg growth of NS modified viruses is due toIFN-mediated effects. The following evidence supports the role ofIFN-mediated effects: (a) The levels of viral protein expression aresimilar in delNS1 virus-infected Vero and in IFNαR−/− cells, but thatthey are markedly reduced in MDCK cells. It should be noted thatIFNαR−/− cells and Vero cells are both deficient in inducing anantiviral IFN response, although the genetic defect responsible for thisdeficiency is different for these two cell lines. (b) Infection with theNS modified virus but not with wild-type virus induced transactivationof an IFN-stimulated reporter gene in 293 cells. (c) Finally, the delNS1virus was able to replicate and to induce disease in mice that weredeficient in IFN signaling, i.e. STAT1−/− animals, but the virus wasnonpathogenic in wild-type mice.

[0028] The importance of type I IFN is illustrated by the fact that manyviruses express antagonists which counteract IFN-mediated responses bythe host. Examples include VA RNAs of adenoviruses, the Epstein-Barrvirus-encoded structural small RNAs, the K3L and E3L gene products ofvaccinia virus, the NSP3 gene product of group C rotavirus, and thereovirus σ3 protein, among others. It is interesting that several ofthese viral products, like the NS1 protein of influenza A virus, areable to bind to dsRNA preventing activation of PKR. Thus, the attenuatedviruses of the present invention may also be used to supplement anyanti-viral therapeutic in that it enhances the IFN-mediated response, aresponse that most viruses have developed complex mechanisms to bypass.

[0029] The attenuated influenza virus of the present invention may beused to express heterologous sequences, including viral and tumorantigens. Thus, the attenuated viruses may be used to express a widevariety of antigenic epitopes, i.e., epitopes that induce a protectiveimmune response to any of a variety of pathogens, or antigens that bindneutralizing antibodies may be expressed by or as part of the chimericviruses. The attenuated virus of the present invention is an excellentvehicle to introduce antigenic epitopes given that it induces anIFN-mediated response and it is not pathogenic to the host.

[0030] In accordance with the present invention, the geneticmanipulation of the NS gene of influenza A viruses may help ingenerating viral vaccine vectors which express novel antigens and/orpolypeptides. Since the NS RNA segment is the shortest among the eightviral RNAs, it is possible that the NS RNA will tolerate longerinsertions of heterologous sequences than other viral RNAs. Moreover,the NS RNA segment directs the synthesis of high levels of protein ininfected cells, suggesting that it would be an ideal segment forinsertions of foreign antigens. However, in accordance with the presentinvention any one of the eight segments of influenza may be used for theinsertion of heterologous sequences.

[0031] Heterologous gene coding sequences flanked by the complement ofthe viral polymerase binding site/promoter, e.g, the complement of3′-influenza virus terminus, or the complements of both the 3′- and5′-influenza virus termini may be constructed using techniques known inthe art. Recombinant DNA molecules containing these hybrid sequences canbe cloned and transcribed by a DNA-directed RNA polymerase, such asbacteriophage T7, T3 or the Sp6 polymerase and the like, to produce therecombinant RNA templates which possess the appropriate viral sequencesthat allow for viral polymerase recognition and activity.

[0032] One approach for constructing these hybrid molecules is to insertthe heterologous coding sequence into a DNA complement of an influenzavirus genomic segment so that the heterologous sequence is flanked bythe viral sequences required for viral polymerase activity; i.e., theviral polymerase binding site/promoter, hereinafter referred to as theviral polymerase binding site. In an alternative approach,oligonucleotides encoding the viral polymerase binding site, e.g., thecomplement of the 3′-terminus or both termini of the virus genomicsegments can be ligated to the heterologous coding sequence to constructthe hybrid molecule. The placement of a foreign gene or segment of aforeign gene within a target sequence was formerly dictated by thepresence of appropriate restriction enzyme sites within the targetsequence. However, recent advances in molecular biology have lessenedthis problem greatly. Restriction enzyme sites can readily be placedanywhere within a target sequence through the use of site-directedmutagenesis (e.g., see, for example, the techniques described by Kunkel,1985, Proc. Natl. Acad. Sci. U.S.A. 82;488). Variations in polymerasechain reaction (PCR) technology, described infra, also allow for thespecific insertion of sequences (i.e., restriction enzyme sites) andallow for the facile construction of hybrid molecules. Alternatively,PCR reactions could be used to prepare recombinant templates without theneed of cloning. For example, PCR reactions could be used to preparedouble-stranded DNA molecules containing a DNA-directed RNA polymerasepromoter (e., bacteriophase T3, T7 or Sp6) and the hybrid sequencecontaining the heterologous gene and the influenza viral polymerasebinding site. RNA templates could then be transcribed directly from thisrecombinant DNA. In yet another embodiment, the recombinant RNAtemplates may be prepared by ligating RNAs specifying the negativepolarity of the heterologous gene and the viral polymerase binding siteusing an RNA ligase. Sequence requirements for viral polymerase activityand constructs which may be used in accordance with the invention aredescribed in the subsections below.

5.2. Generation of Attenuated Viruses

[0033] The present invention relates to genetically engineeredattenuated viruses, and methods for their production. In particular, theinvention relates to attenuated influenza viruses which have beenmodified in such a way to result in an IFN-independent and IFN-inducingphenotype. The following section describes the various approaches whichmay be used in accordance with the invention to generate an attenuatedphenotype. Recombinant DNA techniques can be utilized to engineer sitespecific mutations into one or more noncoding regions of the viralgenome which result in the down-regulation of one or more viral gene.Alternatively, recombinant DNA techniques can be used to engineer amutation, including but not limited to an insertion, deletion, orsubstitution of an amino acid residue(s) or an epitope(s) into a codingregion of the viral genome so that altered or chimeric viral proteinsare expressed by the engineered virus. The invention is based, in part,on the discovery that the down regulation of a viral gene in segmentedviruses results in the production of defective particles at each roundof replication, so that the virus demonstrates attenuatedcharacteristics. In non-segmented viruses, the down-regulation of aviral gene may result in the production of fewer progeny virions thanwould be generated by the corresponding wild type virus. The alterationsof the viral proteins described also result in attenuation for reasonswhich are less well understood.

[0034] Many methods may be used to introduce the live attenuated virusformulations to a human or animal subject to induce an immune response;these include, but are not limited to, oral, intradermal, intramuscular,intraperitoneal, intravenous, subcutaneous and intranasal routes. It ispreferable to introduce the chimeric virus vaccine via its natural routeof infection.

[0035] Any virus may be engineered in accordance with the invention toproduce an attenuated strain suitable for use as a safe live-virusvaccine, including but not limited to viruses belonging to the familiesset forth in Table I below. TABLE I FAMILIES OF HUMAN AND ANIMAL VIRUSESVIRUS CHARACTERISTICS VIRUS FAMILY dsDNA Enveloped PoxviridaeIrididoviridae Herpesviridae Nonenveloped Adenoviridae PapovaviridaeHepadnaviridae ssDNA Nonenveloped Parvoviridae dsRNA NonenvelopedReoviridae Birnaviridae ssRNA Enveloped Positive-Sense Genome No DNAStep in Replication Togaviridae Flaviviridae Coronaviridae Hepatitis CVirus DNA Step in Replication Retroviridae Negative-Sense GenomeNon-Segmented Genome Paramyxoviridae Rhabdoviridae Filoviridae SegmentedGenome Orthomyxoviridae Bunyaviridae Arenaviridae NonenvelopedPicornaviridae Calciviridae # genomes that are composed of nucleotidesequences complementary to the positive-sense strand.

[0036] DNA viruses (e.g., vaccinia, adenoviruses, baculovirus) andpositive strand RNA viruses (e.g., poliovirus) may be readily engineeredusing recombinant DNA techniques which are well known in the art (e.g.,see U.S. Pat. No. 4,769,330 to Paoletti; U.S. Pat. No. 4,215,051 toSmith; Racaniello et al., 1981, Science 214: 916-919). Until recently,however, negative strand RNA viruses (e.g., influenza) were not amenableto site specific genetic manipulation because the viral RNAs are notinfectious. However, a recently developed technique, called “reversegenetics,” allows the engineering and production of recombinant negativestrand RNA viruses.

[0037] The reverse genetics technique involves the preparation ofsynthetic recombinant viral RNAs that contain the non-coding regions ofthe negative strand virus which are essential for the recognition ofviral RNA by viral polymerases and for packaging signals necessary togenerate a mature virion. The recombinant RNAs are synthesized from arecombinant DNA template and reconstituted in vitro with purified viralpolymerase complex to form recombinant ribonucleoproteins (RNPs) whichcan be used to transfect cells. A more efficient transfection isachieved if the viral polymerase proteins are present during in vitrotranscription of the synthetic RNAs. The synthetic recombinant RNPs canbe rescued into infectious virus particles. The foregoing techniques aredescribed in U.S. Pat. No. 5,166,057, issued Nov. 24, 1992 and in Enami& Palese, 1991, J. Virol. 65: 2711-2713, each of which is incorporatedby reference herein in its entirety), and influenza A viruses containinginsertions, deletions and mutations with the stalk portion of the NSgene, which changes acts as a host range mutant.

[0038] 5.2.1. Down-Regulation of Viral Genes

[0039] In accordance with the invention, a non-coding regulatory regionof a virus can be altered to down-regulate a viral gene involved indown-regulating the cellular IFN-mediated response, e.g. reducetranscription of its mRNA and/or reduce replication of vRNA (viral RNA),so that an attenuated virus with an IFN-inducing phenotype is produced.

[0040] This approach, while applicable to any virus, is particularlyattractive for engineering viruses with segmented genomes; i.e., virusesin which the genome is divided into segments that are packaged intovirions. For example, the segmented genome of influenza A virus (anorthomyxovirus) consists of eight molecules of linear negative-sensessRNAs which encode ten polypeptides, including: the RNA-directed RNApolymerase proteins (PB2, PB1 and PA) and nucleoprotein (NP) which formthe nucleocapsid; two surface glycoproteins which project from theenvelope: hemagglutinin (HA) and neuraminidase (NA); and nonstructuralproteins (NS1 and NS2) whose function is unknown. The termini of eachsegment contain the non-coding regions essential for recognition byviral polymerase and for packaging signals necessary to generate amature virion. The sequence of the termini is highly conserved among alleight segments. As another example, the segmented genome of reovirusesconsists of 10 to 12 segments of linear dsRNA which encode 6 to 10 majorstructural polypeptides, a transcriptase and other enzymes.

[0041] The foregoing approach is equally applicable to non-segmented RNAviruses, where the down regulation of transcription of a viral geneinvolved in down-regulating the cellular IFN-mediated response, suchthat it will reduce the production of its mRNA and the encoded geneproduct and result in an interferon-inducing phenotype.

[0042] Any alteration of the regulatory non-coding regions whichdecrease their efficiency or strength may be engineered in accordance inthe invention. For example, the strength of viral promoters can bereduced by alterations in the stem structure. In order to achieve anattenuated phenotype the cis elements of a virus gene involved indown-regulating the cellular IFN-mediated response may be mutated toachieve a dramatic effect on transcription and replication of the gene.

[0043] How influenza A virus packages its eight RNA genome segmentsremains an interesting question. In the past, two different mechanismswere proposed for the packaging of influenza virus RNAs: one suggeststhat the eight RNAs are selectively packaged and the other that viralRNAs are packaged randomly (Compans et al., 1970, In The Biology OfLarge RNA Viruses, Barry & Mahy, Eds., pp. 87-108, Academic Press, N.Y.;Lamb & Choppin, 1983, Ann. Rev. Biochem. 467-506; Smith & Hay, 1982,Virology 118: 96-108). Evidence is now accumulating to support therandom packaging mechanism. The random packaging theory originated fromthe fact that influenza viruses have a low ratio of infectious particlesto physical particles. If one assumes that an average of 11 RNAs arepackaged per virion, the expected ratio is compatible with that found invivo (Enami et al., 1991, Virology 185: 291-298). This model was alsosupported by the finding of a reassortant virus which contained twocopies of the same segment derived from two different viruses(Scholtissek, 1978, Virology 89: 506-516), and further support for thistheory came from a more recent report which described an influenza Avirus which required nine RNAs in order to be infectious (Enami et al.,1991, Virology 185: 291-298).

[0044] In summary, an attenuated phenotype may be achieved by targetingthe cis elements of the NS gene segment to result in down regulation ofthe gene segment. Since the proteins of this virus are unaltered ascompared to wild type virus, attenuation must be the result ofinefficient cis-acting signals. This principal of attenuation may beapplied analogously to other viruses with segmented genomes. Forexample, the introduction of modifications into the noncoding sequencesof rotavirus genes or of genes of other segmented dsRNA viruses (Roneret al., 1990, Virology 179: 845-852) should also allow the pathogenicityof these viruses to be altered.

5.2.2. Alteration of Viral Proteins

[0045] An alternative way to engineer attenuated viruses involves theintroduction of an alteration, including but not limited to aninsertion, deletion or substitution of one or more amino acid residuesand/or epitopes into one or more of the viral proteins involved indown-regulating the cellular IFN-mediated response. This may be readilyaccomplished by engineering the appropriate alteration into thecorresponding viral gene sequence. Any change that alters the activityof the viral protein involved in down-regulating the cellularIFN-mediated response so that viral replication is modified or reducedmay be accomplished in accordance with the invention.

[0046] For example, alterations that interfere with but do notcompletely abolish viral attachment to host cell receptors and ensuinginfection can be engineered into viral surface antigens or viralproteases involved in processing to produce an attenuated strain.According to this embodiment, viral surface antigens can be modified tocontain insertions, substitution or deletions of one or more amino acidsor epitopes that interfere with or reduce the binding affinity of theviral antigen for the host cell receptors. This approach offers an addedadvantage in that a chimeric virus which expresses a foreign epitope maybe produced which also demonstrates attenuated characteristics. Suchviruses are ideal candidates for use as live recombinant vaccines. Forexample, heterologous gene sequences that can be engineered into thechimeric viruses of the invention include, but are not limited to,epitopes of human immunodeficiency virus (HIV) such as gp120; hepatitisB virus surface antigen (HBsAg); the glycoproteins of herpes virus(e.g., gD, gE); VP1 of poliovirus; and antigenic determinants ofnonviral pathogens such as bacteria and parasites to name but a few.

[0047] In this regard, influenza is an ideal system in which to engineerforeign epitopes, because the ability to select from thousands ofinfluenza virus variants for constructing chimeric viruses obviates theproblem of host resistance or immune tolerance encountered when usingother virus vectors such as vaccinia. In addition, since influenzastimulates a vigorous secretory and cytotoxic T cell response, thepresentation of foreign epitopes in the influenza background may alsoprovide for the secretory immunity and cell-mediated immunity. By way ofexample, the insertion, deletion or substitution of amino acid residuesin the HA protein of influenza can be engineered to produce anattenuated strain. In this regard, alterations to the B region or Bregion of HA may be utilized. In accordance with this approach, themalarial epitope (ME 1) of Plasmodium yoelii (NEDSYVPSAEQI) wasintroduced into the antigenic site E of the hemagglutinin of influenza.The resulting chimeric virus has a 500- to 1,000-fold lower LD₅₀ (lethaldose 50) than that of wild type virus when assayed in mice. In anotherembodiment, the major antigenic determinant of poliovirus type 1, i.e.,the BC loop of the VPl of poliovirus type 1 (PASTTNKDKL) was engineeredinto the B region of the influenza HA protein. This chimeric virus isalso attenuated.

[0048] In another embodiment, alterations of viral proteases requiredfor processing viral proteins can be engineered to produce attenuation.Alterations which affect enzyme activity and render the enzyme lessefficient in processing, should affect viral infectivity, packaging,and/or release to produce an attenuated virus. For example, alterationsto the NS protein of influenza can be engineered to reduce NS enzymeactivity and decrease the number and/or infectivity of progeny virusreleased during replication.

[0049] In another embodiment, viral enzymes involved in viralreplication and transcription of viral genes, e.g., viral polymerases,replicases, helicases, etc. may be altered so that the enzyme is lessefficient or active. Reduction in such enzyme activity may result in theproduction of fewer progeny genomes and/or viral transcripts so thatfewer infectious particles are produced during replication.

[0050] The alterations engineered into any of the viral enzymes includebut are not limited to insertions, deletions and substitutions in theamino acid sequence of the active site of the molecule. For example, thebinding site of the enzyme could be altered so that its binding affinityfor substrate is reduced, and as a result, the enzyme is less specificand/or efficient. For example, a target of choice is the viralpolymerase complex since temperature sensitive mutations exist in allpolymerase proteins. Thus, changes introduced into the amino acidpositions associated with such temperature sensitivity can be engineeredinto the viral polymerase gene so that an attenuated strain is produced.

[0051] 5.3. Host-Restriction Based Selection System

[0052] The present invention relates to a host-restriction basedselection system for the identification of genetically manipulatedinfluenza viruses. The selection system of the present invention is moreparticularly related to the identification of genetically manipulatedinfluenza viruses which contain modified NS gene segments. The selectionsystem of the present invention allows for the screening of thegenetically engineered influenza viruses to identify those viruses witha modified NS gene segment.

[0053] The selection system of the present invention is based, in part,on the Applicants' discovery that an engineered influenza A virusdeleted of the NS1 gene was able to grow in a cell line deficient in IFNproduction, whereas the same cell line would not support infection andgrowth of wild type influenza virus. The NS1 deleted virus was unable toinfect and grow in the conventional substrates for influenza virus.Thus, the invention provides a very simple and easy screen to identifythose genetically engineered influenza viruses that contain a modifiedNS1 gene.

[0054] 5.4. Vaccine Formulations Using the Chimeric Viruses

[0055] Virtually any heterologous gene sequence may be constructed intothe chimeric viruses of the invention for use in vaccines. Preferably,epitopes that induce a protective immune response to any of a variety ofpathogens, or antigens that bind neutralizing antibodies may beexpressed by or as part of the chimeric viruses. For example,heterologous gene sequences that can be constructed into the chimericviruses of the invention for use in vaccines include but are not limitedto epitopes of human immunodeficiency virus (HIV) such as gp120;hepatitis B virus surface antigen (HBsAg); the glycoproteins of herpesvirus (e.g. gD, gE); VP1 of poliovirus; antigenic determinants ofnon-viral pathogens such as bacteria and parasites, to name but a few.In another embodiment, all or portions of immunoglobulin genes may beexpressed. For example, variable regions of anti-idiotypicimmunoglobulins that mimic such epitopes may be constructed into thechimeric viruses of the invention.

[0056] Either a live recombinant viral vaccine or an inactivatedrecombinant viral vaccine can be formulated. A live vaccine may bepreferred because multiplication in the host leads to a prolongedstimulus of similar kind and magnitude to that occurring in naturalinfections, and therefore, confers substantial, long-lasting immunity.Production of such live recombinant virus vaccine formulations may beaccomplished using conventional methods involving propagation of thevirus in cell culture or in the allantois of the chick embryo followedby purification.

[0057] In this regard, the use of genetically engineered influenza virus(vectors) for vaccine purposes may require the presence of attenuationcharacteristics in these strains. Current live virus vaccine candidatesfor use in humans are either cold adapted, temperature sensitive, orpassaged so that they derive several (six) genes from avian viruses,which results in attenuation. The introduction of appropriate mutations(e.g., deletions) into the templates used for transfection may providethe novel viruses with attenuation characteristics. For example,specific missense mutations which are associated with temperaturesensitivity or cold adaption can be made into deletion mutations. Thesemutations should be more stable than the point mutations associated withcold or temperature sensitive mutants and reversion frequencies shouldbe extremely low.

[0058] Alternatively, chimeric viruses with “suicide” characteristicsmay be constructed. Such viruses would go through only one or a fewrounds of replication in the host. For example, cleavage of the HA isnecessary to allow for reinitiation of replication. Therefore, changesin the HA cleavage site may produce a virus that replicates in anappropriate cell system but not in the human host. When used as avaccine, the recombinant virus would go through a single replicationcycle and induce a sufficient level of immune response but it would notgo further in the human host and cause disease. Recombinant viruseslacking one or more of the essential influenza virus genes would not beable to undergo successive rounds of replication. Such defective virusescan be produced by co-transfecting reconstituted RNPs lacking a specificgene(s) into cell lines which permanently express this gene(s). Viruseslacking an essential gene(s) will be replicated in these cell lines butwhen administered to the human host will not be able to complete a roundof replication. Such preparations may transcribe and translate—in thisabortive cycle—a sufficient number of genes to induce an immuneresponse. Alternatively, larger quantities of the strains could beadministered, so that these preparations serve as inactivated (killed)virus vaccines. For inactivated vaccines, it is preferred that theheterologous gene product be expressed as a viral component, so that thegene product is associated with the virion. The advantage of suchpreparations is that they contain native proteins and do not undergoinactivation by treatment with formalin or other agents used in themanufacturing of killed virus vaccines.

[0059] In another embodiment of this aspect of the invention,inactivated vaccine formulations may be prepared using conventionaltechniques to “kill” the chimeric viruses. Inactivated vaccines are“dead” in the sense that their infectivity has been destroyed. Ideally,the infectivity of the virus is destroyed without affecting itsimmunogenicity. In order to prepare inactivated vaccines, the chimericvirus may be grown in cell culture or in the allantois of the chickembryo, purified by zonal ultracentrifugation, inactivated byformaldehyde or β-propiolactone, and pooled. The resulting vaccine isusually inoculated intramuscularly.

[0060] Inactivated viruses may be formulated with a suitable adjuvant inorder to enhance the immunological response. Such adjuvants may includebut are not limited to mineral gels, e.g., aluminum hydroxide; surfaceactive substances such as lysolecithin, pluronic polyols, polyanions;peptides; oil emulsions; and potentially useful human adjuvants such asBCG and Corynebacterium parvum.

[0061] Many methods may be used to introduce the vaccine formulationsdescribed above, these include but are not limited to oral, intradermal,intramuscular, intraperitoneal, intravenous, subcutaneous, andintranasal routes. It may be preferable to introduce the chimeric virusvaccine formulation via the natural route of infection of the pathogenfor which the vaccine is designed. Where a live chimeric virus vaccinepreparation is used, it may be preferable to introduce the formulationvia the natural route of infection for influenza virus. The ability ofinfluenza virus to induce a vigorous secretory and cellular immuneresponse can be used advantageously. For example, infection of therespiratory tract by chimeric influenza viruses may induce a strongsecretory immune response, for example in the urogenital system, withconcomitant protection against a particular disease causing agent.

6. MATERIALS AND METHODS

[0062] The following materials and methods were used in the followingSections 7 through 11.

[0063] Viruses and cells. Influenza A/PR/8/34 (PR8) virus was propagatedin 10-day-oldembryonated chicken eggs at 37° C. Influenza A virus 25A-1,a reassortant virus containing the NS segment form the cold-adaptedstrain A/Leningrad/134/47/57 and the remaining genes from PR8 virus(Egorov et al., 1994, Vopr. Virusol. 39:201-205; Shaw et al., 1996, inOptions for the control of influenza III, eds. Brown, Hampson Webster(Elsevier Science) pp. 433-436) was grown in Vero cells at 34° C. The25A-1 virus is ts in mammalian cells, and was used as helper virus forthe rescue of the delNS1 transfectant virus. Vero cells and MDCK cellsin minimal essential medium (MEM) containing 1 μg/ml of trypsin (DifcoLaboratories, Detroit, Mich.) were used for influenza virus growth. Verocells were also used for selection, plaque purification and titration ofthe delNS1 virus. MDCK cells, 293 cells and mouse embryo fibroblasts(MEF) derived from 14-16 day embryos of IFNαR−/− mice were maintained inDMEM (Dulbecco's minimal essential medium) containing 10%heat-inactivated detal calf serum. Immortalized IFNαR−/− fibroblastswere derived from MEF by continuous passage (Todaro et al., 1963, J.Cell. Biol. 17:299-313). Vero cells were grown in AIM-V medium (LifeTechnologies, Grand Island, N.Y.).

[0064] Mice. C57BL/6 mice homozygous for a targeted deletion of STAT1were generated as previously described (Durbin et al., 1996, Cell84:443-450). IFNαR−/− mice have also been described (Hwang et al., 1995,Proc. Natl. Aced. Sci. USA 92:11284-11288). Specific pathogen freeC57BL/6 and BALB/c (wild type) mice were purchased from Taconic Farms.

[0065] Animal infections. Female mice were used for influenza virusinfection at 6 to 12 weeks of age. Intranasal (i.n.) inoculations wereperformed in wild type and STAT1−/− mice under ether anesthesia using 50μl of MEM containing 5×10⁴ plaque forming units (pfu) of delNS1 virus.Animals were monitored daily, and sacrificed when observed in extremis.All procedures were in accord with NIH guidelines on care and use oflaboratory animals.

[0066] Plasmids. pT3delNS1 was made as follows. First, pPUC19-T3/NS PR8,containing the complete NS gene of PR8 virus flanked by the T3 RNApolymerase promoter and BpuAI restriction site was amplified by inversePCR (Ochman et al., 1988, Genetics 120:621-623) using primers5′-CTGAAAGCTTGACACAGTGTTTG-3′ and 5′-GACATACTGCTGAGGATGTC-3′ (CODONGenetic Systems, Weiden, Austria). The obtained cDNA thus lacking theNS1 gene was phosphorylated, Klenow treated, self-ligated and propagatedin E. coli strain TG1. The construct obtained after purification wasnamed pT3delNS1 and verified by sequencing. Plasmids for expression ofthe NP, PB1, PB2, and PA proteins of PR8 virus (pHMG-NP, pHMG-PB1,pHMG-PB2, and pHMG-PA) were previously described (Pleschka et al., 1996,J. Virol. 70:4188-4192). pPOLI-NS-RB was made by substituting the CATopen reading frame of pPOLI-CAT-RT (Pleschka et al., 1996, J. Virol.70:4188-4192) within RT-PCR product derived from the coding region ofthe NS gene of influenza A/WSN/33 (WSN) virus. This plasmid expressesthe NS-specific viral RNA segment of WSN virus under the control of atruncated human polymerase I promoter. pHISG54-1-CAT (Bluyssen et al.,1994. Eur. J. Biochem. 220:395-402) encodes the CAT reporter gene underthe transcriptional control of the IFNα-stimulated promoter of theISG-54K gene.

[0067] Generation of transfectant viruses. Generation of delNS1 viruswas performed by ribonucleoprotein (RNP) transfection (Luytijes et al.,1989, Cell 59:1107-1113). The RNPs were formed by T3 RNA polymerasetranscription from pT3delNS1 linearized with BpUAI in the presence ofpurified nucleoprotein and polymerase of influenza 25A-1 virus (Enami etal., 1991, J. Virol 65:2711-2713). RNP complexes were transfected intoVero cells which were previously infected with 25A-1 virus. Transfectedcells were incubated for 18 hours at 37° C., and the supernatant waspassaged twice in Vero cells at 40° C. and plaque purified three timesin Vero cells covered with agar overlay media at 37° C. The isolateddelNS1 virus was analyzed by RT-PCR using primers5′-GGCCTCTAGATAATACGACTC-ACTATAAGCAAAAGCAGGGTGACAAAG-3′ (complementaryto position 1 to 21 at the 3′ noncoding end of the NS gene) and5′-GATCGCCTTCTATTAGTAGAAA-CAAGGGTGTTTTTATTAAATAAGCTG-3′ (containing thelast 38 nucleotides of the 5′ noncoding end of the NS gene). NS/WSNtransfectant virus was generated as follows. Vero cells in 35-mm disheswere transfected with plasmids pHMG-NP, pHMG-PB1,pHMG-PB2, pHMG-PA andpPOLI-NS-RB, as previously described (Pleschka et al., 1996, J. Virol.70:4188-4192). 2 days postransfection, cells were infected with 5×10⁴pfu of delNS1 virus and incubated 2 more days at 37° C. Cell supernatantwas passaged once in MDCK cells and twice in chicken embryonated eggs.Transfectant viruses were cloned by limiting dilution in eggs. GenomicRNA from purified NS/WSN transfectant virus was analyzed bypolyacrylamide gel electrophoresis, as previously described (zheng etal., 1996, Virology 217:242-251).

[0068] Analysis of virus protein synthesis in infected cells. Cellmonolayers in 35-mm dishes were infected with 2×10⁴ pfu of delNS1 virus.At intervals postinfection, cells were labeled with L-[³⁵S]cysteine andL-[³⁵S] methionine for the indicated times. Labeled cells were lysed in10 mM tris-HCl (pH 7.4) containing 150 mM NaCl, 5 mM EDTA, 1 mM PMSF,10% glycerol, 1% Triton X-100, 1% sodium deoxycholate and 0.1% sodiumdodecylsufate (SDS). Proteins were immunoprecipitated using a rabbitpolyclonal anti-influenza virus serum. Immunoprecipated proteins wereanalyzed by SDS-10% polyacrylamide gel electrophoresis (SDS-PAGE).

[0069] CAT transfections. 293 cell monolayers in 35-mm dishes weretransfected with 1 μg of pHISG54-1-CAT using DOTAP lipofection reagent(Boehringer Mannheim) according to the manufacturer's instructions, andincubated at 37° C. 1 day postransfection cells were infected withdelNS1 virus or PR8 virus at the indicated multiplicities of infection(MOI). As controls, cells were mock-infected or transfected with 50 μgof poly(I-C). After 1 day more at 37° C., cell extracts were made andassayed for CAT activity, as described (Percy et al., 1994, J. Virol.68:4486-4492).

[0070] 7. Example: Generation of the Transfectant Influenza VirusdelNS1, Lacking the NS1 Gene

[0071] The NS-specific viral RNA segment of influenza A virus encodesboth the NS1 and NEP (nuclear export protein) proteins. UnsplicedNS-specific mRNA translates into the NS1 protein, while the spliced RNAdirects the synthesis of the NEP. The plasmid pT3delNS1 was constructedwhich expresses a mutated NS gene from influenza PR8 virus. This mutatedRNA segment contains a deletion of the NS1-specific open reading frame(nt positions 57 to 528 of the PR8 NS gene (Baez et al., 1980, NucleicAcids Res. 8:5845-5858) and thus it encodes only the NEP (FIG. 1). RNPtransfection of the delNS1 gene using the ts 25A-1 helper virus yieldeda progeny virus which was able to grow at 40° C. in Vero cells.Amplification of the NS gene of the rescued virus by RT-PCR confirmedthe substitution of the NS gene of the helper virus with that derivedfrom the transfected delNS1 gene (FIG. 2).

[0072] 8. Example: Growth Properties of delNS1 Virus in Tissue Cultureand Eggs

[0073] The growth properties of delNS1 virus and wild-type PR8 viruswere compared in Vero cells, MDCK cells, and 10-day-old embryonatedchicken eggs. Cell monolayers containing approximately 106 Vero or MDCKcells were infected with delNS1 virus or PR8 virus at an MOI ofapproximately 0.0005. After 4 days incubation at 37° C. using MEMcontaining 1 μg/ml of trypsin, supernatants were used in ahemagglutination assay. Alternatively, the allantoic cavity of10-day-old embryonated chicken eggs was injected with 10⁴ pfu of delNS1or PR8 virus, and the hemagglutination titer in the allantoic fluids wasdetermined after 3 days of incubation of 37° C. As shown in Table 2,delNS1 virus was able to grow in Vero cells to titers of 16 as comparedto a tier of 128 for wild-type PR8 virus. However, delNS1 virusreplication was severely impaired in MDCK cells and in eggs(hemagglutination titers were undetectable in these samples). TABLE 2DelNS1 virus replication in tissue culture cells and eggsHemagglutination titer¹ Culture media delNS1 WT PR8² Vero cells 16 128MDCK cells <2 512 Eggs <2 2,048

8.1. Deletion of the NS1 Gene is Responsible for the Growth Propertiesof delNS1

[0074] In order to prove that the impaired viral growth of delNS1 virusin MDCK cells and eggs was due to the deletion of the NS1 gene and notto possible differences in other RNA segments of delNS1 and PR8 viruses,we used delNS1 virus as helper virus to rescue a wild-type NS gene.Transfections were carried as described in Materials and Methods using aplasmid-based expression system (Pleschka et al., 1996, J. Virol.70:4188-4192) for the wild-type NS gene of influenza A/WSN/34 virus.Selection of transfectant NS/WSN viruses were done by serial passages inMDCK cells and eggs. SDS-PAGE analyses of purified viral RNA from NS/WSNvirus confirmed the wild-type length of its NS RNA segment. TransfectantNS/WSN virus containing the NS RNA segment derived from WSN virus andthe remaining segments from delNS1 (PR8) virus was able to grow toidentical titers than those of wild-type PR8 virus in MDCK cells and10-day-old embryonated chicken eggs.

[0075] 8.2. Viral Protein Expression Levels in delNS1 Virus-InfectedMDCK and Vero Cells

[0076] In order to investigate if the deficiency of delNS1 virusreplication MDCK cells correlated with a decrease in the expressionlevels of viral proteins, [³⁵S]-labeling experiments were performed.MDCK or Vero cells were infected with delNS1 virus at an MOI of 0.02 andlabeled with L-[³⁵S]cysteine and L-[³⁵S]methionine for the indicatedtimes. After labeling, viral proteins were immunoprecipitated from cellextracts and separated by PAGE (FIG. 3). Quantitation of the ³⁵S signalindicates that approximately 20-fold less viral protein was synthesizedby infected MDCK cells.

[0077] 8.3. DelNS1 Virus-Infected Vero and IFNαR−/− Cells have SimilarLevels of Viral Protein Expression

[0078] The reason for the differences between Vero and MDCK cells insupporting delNS1 virus replication and viral protein expression mayrelate to the inability of Vero cells to synthesize IFN (Desmyter etal., 1968, J. Virol. 2:955-961; Mosca et al., 1986, Mol. Cell. Biol.6:2279-2283; Diaz et al., 1988, Proc. NeH. Acad. Sci. USA 85:52595263).In order to test this hypothesis, the pattern of viral proteinexpression was investigated in a murine cell line which is unable torespond to IFN. IFNαR knock-out cells were infected with delNS1 virus atan MOI of 0.02 and labeled as described above. As a control, delNS1virus-infected Vero cells were labeled from 8 to 10 hours postinfectionat the same time. Levels of viral protein expression were similar inboth cell lines (FIG. 4), suggesting that delNS1 virus is able toreplicate in IFN-deficient systems. It should be noted that we could notinvestigate multicycle replication of delNS1 virus in IFNαR−/− cellsbecause these cells die rapidly in the presence of trypsin, which isrequired for viral hemagglutinin activation and virus infectivity.

[0079] 9. Example: Delta NS1 Virus is a Potent Inducer of InterferonResponses

[0080] Stimulation of transcription from an IFN-regulated promoter byinfection with transfectant delNS1 virus. In order to investigate if thedeletion of the NS1 gene in delNS1 virus results in an enhanced IFNresponse in infected cells we performed transfection experiments usingpHISG54-1-CAT. This plasmid contains the CAT reporter gene in front ofthe IFNα-stimulated promoter of the ISG-54K gene (Bluyssen et al., 1994,Eur. J. Biochem. 220:395-402). 293 cells were transfected withpHISG54-1-CAT and infected with delNS1 virus or wild-type PR8 at theindicated MOIs as described in Material and Methods. CAT activity in theinfected cell extracts was compared with that in uninfected cellextracts. As positive control, transcription from the IFN-regulatedpromoter was stimulated using poly(I-C). As shown in FIG. 5, infectionat an MOI of 0.05 with delNS1 virus, but not with wild-type virus,induced approximately a 6-fold stimulation of reporter gene expression.The transfectant virus lacking the NS1 gene was thus impaired in itsability to inhibit the IFN response in 293 infected cells.

10. Example: Pathogenicity of Delta NS1 Virus

[0081] Transfectant delNS1 virus is pathogenic in STAT1−/− mice. Theability of delNS1 virus to replicate and cause disease in wild-type miceand in mice deficient in the IFN responses was investigated. For thispurpose, 5×10⁴ pfu of delNS1 virus was used to i.n. infect three C57BL/6mutant mice which were homozygous for a targeted deletion of STAT1, atransactivator which is required for the IFN signaling (Durbin et al.,1996, Cell 84:443-450; Merez et al., 1996, Cell84:431-442). Three tofour wild-type C57BL/6 and BALB/c mice were also inoculated with delNS1virus. Infected-STAT1−/− mice looked sick by day 3 postinfection. By day7 postinfection, all three infected STAT1−/− mice died (Table 3). DelNS1virus was recovered from the lungs of STAT1−/− dying mice, indicatingthat the virus was replicating in these animals. However, all wild-typeinfected mice survived infection with delNS1 without developing anysymptoms of disease (Table 3). TABLE 3 Survival of mice following delNS1virus infection¹ Day postinfection Mice 1 day 7 day 14 day STAT1−/−C57BL/6 3 of 3 0 of 3 0 of 3 Wild-type C57BL/6 3 of 3 3 of 3 3 of 3Wild-type BALB/c 4 of 4 4 of 4 4 of 4

[0082] 11. Example: Preinoculation with delNS1 Virus InhibitsReplication of Influenza

[0083] In order to investigate if preinoculation with the delNS1 virushas an inhibitory effect on infection or replication of wild-typeinfluenza, the following experiment was conducted.

[0084] In this study 10-day old embroyated chicken eggs were inoculatedwith 20,000 pfu of the delNS1 virus or with PBS into the allantoiccavity. After 8 hours of incubation at 37° C., the eggs were reinfectedwith 10³ pfu of H1N1 influenza A/WSN/33 (WSN); H1N1 influenza A/PR/8virus (PR8), H3N2 influenza A/X-31 virus (X-31), influenza B/Lee/40virus (B-Lee) or Sendai virus and incubated for an additional 40 hoursat 37° C., except for B-Lee infected cells which were incubated at 35°C. As shown in FIG. 6, the delNS1 virus treated eggs resulted inundetectable levels of viral infection, when compared to the untreatedcells. Thus, demonstrating the anti-viral activity of the delNS1 virusand its potential as an anti-viral therapeutic and prophylactic.

[0085] The present invention is not to be limited in scope by thespecific-embodiments described which are intended as singleillustrations of individual aspects of the invention, and any constructsor viruses which are functionally equivalent are within the scope ofthis invention. Indeed, various modifications of the invention inaddition to those shown and described herein will become apparent tothose skilled in the art from the foregoing description and accompanyingdrawings. Such modifications are intended to fall within the scope ofthe appended claims.

[0086] Various references are cited herein, the disclosures of which areincorporated by reference in their entireties.

What is claimed is:
 1. An attenuated genetically engineered influenza Avirus with an interferon-inducing phenotype containing a knockout of theNS1 gene segment.
 2. An attenuated genetically engineered influenza Avirus containing a deletion of the entire NS1 gene segment which iscapable of infecting and replicating in an interferon (IFN) deficientcell line.
 3. The attenuated virus of claim 1 or 2 which further encodesa heterologous sequence.
 4. The attenuated virus of claim 1 or 2 inwhich the heterologous sequence encodes a viral antigenic peptide. 5.The attenuated virus of claim 1 or 2 in which the heterologous sequenceencodes a tumor antigenic peptide.
 6. A vaccine comprising an attenuatedgenetically engineered influenza A virus containing a complete deletionin its NS1 gene segment in a suitable pharmaceutical formulation.
 7. Thevaccine of claim 6 which is formulated for intranasal delivery.
 8. Amethod of vaccination comprising administering to a subject the vaccineof claim 6.